This recent paper in PLoS Genetics by Cheryl C. Y. Li and colleagues (from a group that did earlier work with the agouti mouse model) was really fascinating- they looked at changes in methylation variability in response to dietary supplementation of methyl donors across multiple generations of mice. Some very novel and important findings (summary at the bottom).

This time, they focused on CpG islands in the whole genome of (C57BL/6) mice fed methyl donors (similar to agouti studies: choline, betaine, L-methionine, ZnSO4, folic acid, B12) before and during pregnancy and through lactation. Isogenic mice without methyl donor consumption (but otherwise same diet) was the control group.

Hepatocytes were sequenced in the control mice and after the 1st and 6th generations of the supplemented mice.

Interestingly, they found that global cytosine methylation via HPLC from the hepatocytes was not increased from methyl donor supplementation.

To measure differences at each loci (other than agouti (Avy) and AxinFu), they examined the CpG islands with DNA microarrays. From this they found that methylation pattern variability (at each locus) was less (but still apparent) between datasets from control animals than between sets from the supplemented mice. So even between animals on the same diets without added methyl donors, epigenetic variation exists. This isn’t novel (see this post & others), but still important to verify.

Not only was methylation pattern variability greater in supplemented mice, but in this group from generation 1 compared to generation 6, pattern variability and spatial distance increased (suggesting more distinct patterns as well; this was true compared to controls too). Likewise, generation 6 supplemented mice had greater variability compared to the controls than generation 1 compared to the controls.

Together, their results suggest that mice supplemented with methyl donors have CpG island loci with more or less methylation compared to control mice, and that these patterns increase in variability and difference over generations.

The researchers then identified the loci with the greatest within group variability (standard deviation above 95th percentile); 2110 were in the control group, 2606 in 1st generation supplemented mice, and 3640 in 6th generation supplemented. The 6th generation supplemented mice had the most unique variable loci, followed by the 1st generation, then the controls. Thus, the number of loci that display variability increases with each generation of supplementation with methyl donors, and this happens stochastically.

Finally, some of these loci with the most methylation variability were more common between groups than others, so the authors performed gene ontology analyses to see if they had common functions. They found overlap in genes involved in transcription, development, and organogenesis. So it isn’t random- some genes are more likely to be methylated than others.

Summary

In summary, sustained supplementation of methyl donors to mice over generations stochastically increases the methylation pattern variability. This demonstrates an increased epigenetic plasticity over time, which could help to better adapt to environmental changes. This is how they say it:

That the effect becomes more pronounced with multigenerational exposure suggests that at least some of the induced changes are heritable. If so, phenotypic diversity created by an environmentally-induced increase in epigenetic variability might be acted upon by natural selection independently of genotype (Figure 5). This could enable rapid (within a few generations) adaptation to new environments [35]–[37], and because no genetic change is required, the acquired phenotypes would potentially be reversible if environmental conditions reverted. A sustained environmental change over a longer period might eventually result in a permanent epigenetic change which can in turn facilitate genetic mutation through the increased mutability of 5-methylcytosine [32], [38]–[39].

Figure 5:

Another important quote:

It is also possible that small, widespread changes in methylation induced by a poor intrauterine environment may become magnified over a lifetime and hence accelerate age-associated epigenetic decline [15]; this may go some way to explaining why fetal programming effects are observed later in life.

Even though the most variably methylated genes here overlap in ontologies, they report that recent evidence suggests small, subtle methylation changes throughout the genome, instead of “at a few key regulatory genes” occurs in response to nutritional changes. This is in contrast to a recent study by Carone et al. that I wrote about here that suggested an increased methylation near PPARalpha seemed to have mediated many of the downstream changes in gene expressions in response to protein restriction. They found a larger change (~30%) in methylation than what happens in most studies (<10%), so maybe a little of both is possible?

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